Detection of enhanced multiplex signals by surface enhanced raman spectroscopy (sers)

ABSTRACT

Various methods of using Raman-active or SERS-active probe constructs to detect analytes in biological samples, such as the nucleic acid and/or protein-containing analytes in a body fluid are provided.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods and devices useful toidentify the presence of an analyte in a sample.

2. Background Information

Surface-enhanced Raman scattering (SERS) is a sensitive spectroscopicmethod for detection of an analyte. Raman Spectroscopy probesvibrationally excitable levels of an analyte. Once a vibrational levelis excited by a photon, the energy of the photon shifts by an amountequal to that of the level (Raman scattering). A Raman spectrum, similarto an infrared spectrum, consists of a wavelength distribution of bandscorresponding to molecular vibrations specific to the sample beinganalyzed (the analyte). In the practice of Raman spectroscopy, the beamfrom a radiation source is focused upon the sample to thereby generateinelastically scattered radiation, which is optically collected anddirected into a wavelength-dispersive spectrometer in which a detectorconverts the energy of impinging photons to electrical signal intensity.In SERS, analyte molecules are adsorbed on noble metal nanoparticles.These nanoparticles, once excited by light, set up plasmon modes, which,in turn, create near fields around each particle. These fields cancouple to analyte molecules in the near field regions. As a result,concentration of the incident light occurs at close vicinity of thenanoparticles enhancing the Raman scattering from the analyte molecules.This method can enhance the detection of biological systems by as muchas a factor of 10¹⁴.

Multiplexing is a demanding approach for high throughput assays invarious areas such as biological research, clinical diagnosis and drugscreening because of its great potentials in increasing efficiencies ofchemical and biochemical analyses. In a multiplex assay, multiple probesare used that have specificities to corresponding analytes in a samplemixture. One of the critical challenges in establishing a multiplexplatform is to develop a probe identification system that hasdistinguishable components for each individual probe in a large probeset.

Previous methods have utilized SERS in combination with multiplexanalysis. Such methods utilize target-coated gold particles and DNAprobes co-modified with both a Raman dye and thiol group (Cao et al,Science 297:1536). However, such reagents are generally expensive tomanufacture and labor intensive to use. In addition, coupling the Ramandye and thiol group to the analyte does not provide flexibility inapplication and/or removal of the dye or SERS substrate. Thus, thereexists a need for compositions and methods that provide lower costs andincreased flexibility in labeling analytes and capture reagents duringmultiplex analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic flow chart illustrating an improved method fordetection of an analyte. Capture reagents bound to a solid support(e.g., antibodies) include a Raman label. The binding of a targetanalyte is subsequently detected by Raman detection.

FIG. 1B is a schematic flow chart illustrating previous methods fordetection of an analyte absent interchangeable binding members and Ramanlabeled capture reagents.

FIG. 2 is a schematic diagram illustrating an improved method fordetection of an analyte. The exemplary diagram illustrates proteintargets binding to antibodies immobilized to a solid surface and labeledwith Raman labels.

FIG. 3A is a schematic flow chart illustrating an improved method fordetection of a nucleic acid sequence (e.g., SNP). Capture nucleic acidsbound to a solid support include hybridize a target nucleic acidassociated with a first binding member. Hybridization of the targetnucleic acid is subsequently detected by Raman detection.

FIG. 3B is a schematic flow chart illustrating previous methods fordetection of a nucleic acid sequence absent interchangeable bindingmembers.

FIG. 4 is a schematic diagram illustrating an improved method fordetection of a nucleic acid sequence. The exemplary diagram illustratestarget nucleic acids hybridizing to immobilized capture nucleic acidmolecules.

The following detailed description contains numerous specific details inorder to provide a more thorough understanding of the disclosedembodiments of the invention. However, it will be apparent to thoseskilled in the art that the embodiments can be practiced without thesespecific details. In other instances, devices, methods, procedures, andindividual components that are well known in the art have not beendescribed in detail herein.

DETAILED DESCRIPTION OF THE INVENTION

The various embodiments of the invention relate to signal amplificationmethods for multiplex biological assays. In general, biological targetcomplexes are tagged by a seed substance that can catalyze the formationof a surface-enhanced Raman scattering (SERS) substrate. The targetcomplexes can then bind to capture reagents which include a Raman label.The SERS substrate is then generated on the seed substance throughreduction of metal cations. The target signals are detected by SERSmeasurement of the Raman labels. More specifically, embodiments of theinvention provide target analytes functionalized with specific bindingmembers comprising seed particles. Embodiments of the invention furtherrelate to target complexes formed between such target analytes and acapture reagent bound to a solid substrate. The capture reagentoptionally includes a Raman label. Other embodiments of the inventionrelate to methods of detecting binding of a target analyte to a capturereagent coupled with surface-enhanced Raman scattering (SERS)spectroscopy, to perform multiplexed detection of analytes. This isexemplified for polypeptide targets in FIGS. 1A, 1B and 2, and fornucleic acid targets in FIGS. 3A, 3B and 4.

Accordingly, in one embodiment, a biological target complex including atarget analyte associated with a first specific binding member isprovided. The target complex further includes a second specific bindingmember that binds to the first specific binding member forming a targetcomplex. The second specific binding member includes a seed particlesuitable for catalyzing the formation of a surface enhanced Ramanscattering (SERS) substrate. Subsequently, the SERS substrate can beactivated to provide a SERS effect. The complex further includes acapture reagent bound to a solid substrate. The capture reagent caninclude a Raman label.

“Target analyte,” as used herein, is the substance to be detected in thetest sample using the present invention. The analyte can be anysubstance for which there exists a naturally occurring capture reagent(e.g., an antibody, polypeptide, DNA, RNA, cell, virus, etc.) or forwhich a capture reagent can be prepared, and the target analyte can bindto one or more capture reagents in an assay. “Target analyte” alsoincludes any antigenic substances, haptens, antibodies, and combinationsthereof. The target analyte can include a protein, a peptide, an aminoacid, a carbohydrate, a hormone, asteroid, a vitamin, a drug includingthose administered for therapeutic purposes as well as thoseadministered for illicit purposes, a bacterium, a virus, and metabolitesof or antibodies to any of the above substances

“Target analyte-analog”, as used herein, refers to a substance whichcross reacts with an analyte capture reagent although it may do so to agreater or lesser extent than does the target analyte itself. The targetanalyte-analog can include a modified target analyte as well as afragmented or synthetic portion of the target analyte molecule so longas the target analyte analog has at least one epitopic site in commonwith the target analyte of interest.

“Specific binding member,” as used herein, is a member of a specificbinding pair, i.e., two different molecules where one of the molecules(e.g., a first specific binding member), through chemical or physicalmeans, specifically binds to the second molecule (e.g., a secondspecific binding member). In addition to antigen and antibody-specificbinding pairs, other specific binding pairs include biotin and avidin,carbohydrates and lectins, complementary nucleotide sequences (includingprobe and captured nucleic acid sequences used in DNA hybridizationassays to detect a target nucleic acid sequence), complementary peptidesequences, effector and receptor molecules, enzyme cofactors andenzymes, enzyme inhibitors a and enzymes, cells, viruses and the like.Furthermore, specific binding pairs can include members that are analogsof the original specific binding member. For example a derivative orfragment of the analyte, i.e., an analyte-analog, can be used so long asit has at least one epitope in common with the analyte. Immunoreactivespecific binding members include antigens, haptens, antibodies, andcomplexes thereof including those formed by recombinant DNA methods orpeptide synthesis.

“Ancillary Specific binding member,” as used herein, is a specificbinding member used in addition to the specific binding members of thetarget analyte and the capture reagent and becomes a part of the finalcomplex. One or more ancillary specific binding members can be used inan assay. “Binding,” as used herein, is any process resulting in theformation of coupled moieties. The process of “binding” refers to thedirect or indirect attachment of one moiety to another through theformation of at least one bond, which can include covalent, ionic,coordinative, hydrogen, or Van der Waals bonds, or non-chemicalinteractions, for example, hydrophobic interactions. It is understoodthat two moieties can be coupled to each other by numerous ways. Suchcoupling can include, but is not limited to, specific non-covalentaffinity interactions, for example streptavidin: or avidin:biotininteractions and hapten:antibody interactions; hydrophobic interactions;magnetic interactions; polar interactions, for example, “wetting”associations between two polar surfaces or betweenoligonucleotide/polyethylene glycol; formation of a covalent bond, forexample, an amide bond, a disulfide bond, a thioether bond, an etherbond, a carbon-carbon bond; or via other crosslinking agents; or via anacid-labile linker. Exemplary coupled moieties include, but are notlimited to, antibody-epitope complexes, receptor-ligand complexes orcomplementary nucleic acid complexes. Exemplary target analyte-capturereagent complexes include a target nucleic acid sequence (i.e., a targetanalyte) hybridizing to a complementary nucleic acid sequence (i.e. acapture reagent). Other exemplary target analyte-capture reagentcomplexes include a target polypeptide (i.e., a target analyte) bindingto a receptor or antibody (i.e. a capture reagent), thus forming aligand binding pair. The extent of the binding is influenced by thepresence, and the amount present, of the target analyte. “Associated,”as used herein, is the state of two or more molecules and/orparticulates being held in close proximity to one another.

“Capture reagent,” as used herein, is a molecule or compound capable ofbinding the target analyte or target reagent, which can be directly orindirectly attached to a substantially solid material. The capture agentcan be any substance for which there exists a naturally occurring targetanalyte (e.g., an antibody, polypeptide, DNA, RNA, cell, virus, etc.) orfor which a target analyte can be prepared, and the capture reagent canbind to one or more target analytes in an assay.

“Seed particle,” as used herein, is any substance that can precipitateformation of a nanoparticle from a metal colloid solution and supportthe phenomenon of a surface-enhanced Raman light scattering (SERS) orsurface-enhanced resonance Raman light scattering (SERRS). Examples ofseed particles include, but are not limited to: Colloids of gold orsilver, Pt, Cu, Ag/Au, Pt/Au, Cu/Au, coreshell or alloy particles;particles or flakes of gold, silver, copper, or other substancesdisplaying conductance band electrons. As the particle surfaceparticipates in the SERS and SERRS effect, flakes or particles ofsubstances not displaying conductance band electrons, which have beencoated with a substance which does, also become suitable particulates.

“Raman label,” as used herein, is any substance which produces adetectable Raman spectrum, which is distinguishable from the Ramanspectra of other components present, when illuminated with a radiationof the proper wavelength. Other terms for a Raman label include“Raman-active label,” “Raman dye” and “Raman reporter molecule.” A Ramanlabel includes any organic or inorganic molecule, atom, complex orstructure, including but not limited to synthetic molecules, dyes,naturally occurring pigments such as phycoerythrin, organicnanostructures such as C60, buckyballs and carbon nanotubes, metalnanostructures such as gold or silver nanoparticles or nanoprisms andnano-scale semiconductors such as quantum dots. Numerous examples ofRaman labels are disclosed below. Exemplary Raman labels are provided inTable 1 below. The skilled artisan will realize that such examples arenot limiting, and that “Raman label” encompasses any organic orinorganic atom, molecule, compound or structure known in the art thatcan be detected by Raman spectroscopy. A particular type of “Ramanlabel” includes “Raman dyes.” Examples of Raman dyes include chemicallabels such as cresyl fast violet (CFV, Fluka), brilliant cresyl blue(BCB, Allied Chemical and Dye) and p-aminobenzoic acid (PABA, Aldrich).Additional dyes include Cy3, Cy3.5, Cy5, TAMRA (TMR), Texas-Red (TR) andRhodamine 6G (RD).

Raman labels offer the advantage of producing sharp spectral peaks,allowing a greater number of distinguishable labels to be attached toprobes. Additional non-limiting examples of Raman-active labels of useinclude TRIT (tetramethyl rhodamine isothiol), NBD(7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blueviolet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine,biotin, digoxigenin, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, TET (6-carboxy-2′,4,7,7′-tetrachlorofluorescein), HEX(6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein), Joe(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein)5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein,5-carboxy rhodamine, Tamra (tetramethylrhodamine), 6-carboxyrhodamine,Rox (carboxy-X-rhodamine), R6G (Rhodamine 6G), phthalocyanines,azomethines, cyanines (e.g. Cy3, Cy3.5, Cy5), xanthines,succinylfluoresceins, N,N-diethyl-4-(5′-azobenzotriazolyl)-phenylamineand aminoacridine. These and other Raman-active tags can be obtainedfrom commercial sources (e.g., Molecular Probes, Eugene, Oreg.).

It is contemplated that the Raman-active label may comprise one or moredouble bonds, for example carbon to nitrogen double bonds. It is alsocontemplated that the Raman-active labels may comprise a ring structurewith side groups attached to the ring structure, such as polycyclicaromatic compounds in general. Compounds with side groups that increaseRaman intensity include compounds with conjugated ring structures, suchas purines, acridines, Rhodamine dyes and Cyanine dyes. The overallpolarity of a polymeric active molecular Raman code is contemplated tobe hydrophilic, but hydrophobic side groups can be included. Other tagsthat can be of use include cyanide, thiol, chlorine, bromine, methyl,phosphorus and sulfur.

Included herein are guidelines useful for designing and manufacturingRaman labels. Briefly, exemplary Raman labels provided herein encompassthose that comprise any combination of the following attributes: 1) aconjugated aromatic system (generally two or more rings); 2) one or morenitrogen or sulphur atoms with a lone pair of electrons (for Agbinding), preferably two of such atoms on the same side of the moleculeso that they can chelate a metal atom; 3) as few oxygen atoms aspossible; 4) few or no free OH groups in proximity to the Ag bindingsite; and 5) few competing Ag binding modes.

Accordingly, molecules useful for providing metal surface adsorptiongenerally include at least one N or S atoms with a lone pair ofelectrons (for Ag binding). In some aspects, it is useful to have two ofsuch atoms on the same side of the molecule so that they can chelatemetal atoms. Additionally, in other aspects, a positive charge in themolecule, such as N⁺, S⁺, or C⁺, is included.

Generally, silver colloids are stable with a relatively large surfacepotential (−60 mV or lower); when organic compound molecules areadsorbed on to silver colloid surfaces, the potential (zeta potential)is reduced and thus cause colloid agglutination with the organiccompounds as the “glue”. The present study has identified compounds witha conjugated aromatic system that can more efficiently induceaggregation of Ag particles. N or S atoms are suitable for stablebinding to Ag surface, and a single binding mode anchored by twochelating electron-donor atoms aids in generating strong Raman signalswith simple signature. In general, O atoms, especially those from freehydroxyl groups, compete with N and S for Ag surface binding.

Molecules suitable for providing a strong Raman signal generally includethose possessing strong absorption of UV-Visible light (conjugateddouble bonds and aromatic system). Molecules with strong absorption nearthe Raman excitation wavelength are included because of their resonanceeffect. Also included are those molecules with vibration modes such asC—N bond stretching, C—C bond stretching, and 6-member ring breath modesin an aromatic system. Also included are those molecules with few Oatoms because, generally, C—O, O—H, and C═O bonds do not provide strongRaman signals.

Also provided are chemical structures that impart a unique Ramansignature for a Raman label. The Raman shift of a particular mode can be“moved” to either longer wavelength or shorter wavelength based on itschemical structure environment. For example, neighboringelectron-withdrawing group (conjugated aromatic ring, CN, etc.) may movethe Raman peak to higher wave number, and electron-donating groups(Amine, thiol, etc.) do the opposite. In addition, unique Ramansignatures can be imparted to a molecule by avoiding same vibrationmodes occurring at different parts of the molecule unless they aresymmetrical. Such structure gives double peaks or broadened single peak,which generally complicate the Raman signature.

In certain embodiments, the Raman-active labels used in the inventionmethods and complexes can be independently selected from the groupconsisting of nucleic acids, nucleotides, nucleotide analogs, baseanalogs, fluorescent dyes, peptides, amino acids, modified amino acids,organic moieties, quantum dots, carbon nanotubes, fullerenes, metalnanoparticles, electron dense particles and crystalline particles, or acombination of any two or more thereof.

The present invention contemplates the use of any suitable particlehaving Raman labels and specific binding substances attached theretothat are suitable for use in detection assays. In practicing thisinvention, however, nanoparticles are preferred. The size, shape andchemical composition of the particles will contribute to the propertiesof the resulting probe including the DNA barcode. These propertiesinclude optical properties, optoelectronic properties, electrochemicalproperties, electronic properties, stability in various solutions, poreand channel size variation, ability to separate bioactive moleculeswhile acting as a filter, etc. The use of mixtures of particles havingdifferent sizes, shapes and/or chemical compositions, as well as the useof nanoparticles having uniform sizes, shapes and chemical composition,are contemplated. Examples of suitable particles include, withoutlimitation, nano- and microsized core particles, aggregate particles,isotropic (such as spherical particles) and anisotropic particles (suchas non-spherical rods, tetrahedral, prisms) and core-shell particlessuch as the ones described in U.S. patent application Ser. No.10/034,451, filed Dec. 28, 2002 and International application no.PCT/US01/50825, filed Dec. 28, 2002, which are incorporated by referencein their entirety.

Nanoparticles useful in the practice of the invention include metal(e.g. gold, silver, copper and platinum), semiconductor (e.g., CdSe,CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g.,ferromagnetite) colloidal materials. Other nanoparticles useful in thepractice of the invention include ZnS, ZnO, TiO.sub.2, AgI, AgBr,HgI.sub.2, PbS, PbSe, ZnTe, CdTe, In.sub.2S.sub.3, In.sub.2Se.sub.3,Cd.sub.3P.sub.2, Cd.sub.3As.sub.2, InAs, and GaAs. The size of thenanoparticles is preferably from about 1.4 nm to about 150 nm (meandiameter), more preferably from about 5 to about 50 mm, most preferablyfrom about 10 to about 30 nm. The nanoparticles may also be rods,prisms, cubes, tetrahedra, or core shell particles.

“SERS (Surface-Enhanced Raman Scattering)” means the increase in Ramanscattering exhibited by certain molecules in proximity to certain metalsurfaces. “SERRS (Surface Enhanced Resonance Raman Scattering)” resultswhen the adsorbate at a SERS active surface is in resonance with thelaser excitation wavelength. The resultant enhancement is the product ofthe resonance and surface enhancement.

“SERS substrate,” as used herein, includes a stain such as a silver orgold stain that provides for activating Raman labels on particles toproduce a SERS effect. “Stain,” as used herein, includes material, e.g.,gold, silver, etc., that can be used to produce or enhance a detectablechange in any assay described herein. For example, silver staining canbe employed with any type of nanoparticles that catalyze the reductionof silver. Thus, gold colloid exposed to a staining solution containingAgNO₃ can serve as nucleation sites for the deposition of Ag.

“Intermediary molecule,” as used herein, is any substance that includesa specific binding member and binds to a target analyte. Exemplaryintermediary molecules include antibodies attached to a specific bindingmember. As shown in FIG. 2, an exemplary intermediary molecule includessecond antibody comprising nanogold seeds. Such molecules generally bindto the target analyte following binding of the target analyte to theimmobilized capture reagent.

“Radiation,” as used herein, is an energy in the form of electromagneticradiation which, when applied to a test mixture, causes a Raman spectrumto be produced by the Raman-active label therein.

The complexes and methods provided herein are distinguishable fromprevious reports combining SERS detection with Raman label applications.For example, gold nanoparticles combined with surface-enhanced Ramanscattering spectroscopy for detection and identification of single dyemolecules has been described by Cao et al (Science 297:1536). Cao et aldesigned a probe that is built around a 13 nm gold nanoparticle. Thenanoparticles are coated with hydrophilic oligonucleotides containing aRaman dye at one end and terminally capped with a small moleculerecognition element (e.g. biotin). This molecule is catalytically activeand will be coated with silver in the solution of Ag(I) andhydroquinone. After the probe is attached to a small molecule or anantigen it is designed to detect, the substrate is exposed to silver andhydroquinone solution. The silver-plating occurs in close proximity tothe Raman dye, which allows for dye signature detection with a standardRaman microscope. In contrast, the complexes disclosed herein arecomprised of a Raman dye that is separate from, for example, a goldnanoparticle suitable for supporting a SERS substrate. The biologicaltarget complexes comprise a seed particle capable of catalyzing theformation of a SERS substrate. The seed particle is associated with asecond binding member which binds to a first binding member associatedwith a target analyte. Once the target analyte binds to a capturereagent associated with a Raman label the SERS substrate is generatedthrough reduction of metal cations. Alternatively, when a nucleic acidis a the target analyte, the Raman label is added to the SERS substratesubsequent to formation (see e.g., FIGS. 3A, 3B and 4).

Accordingly, in another embodiment, a method for detecting ananalyte-capture reagent complex by Raman spectroscopy is provided. Themethod includes providing a target analyte associated with a firstspecific binding member and providing a capture reagent bound to a solidsubstrate. The capture reagent includes a Raman label. The methodfurther includes contacting the target analyte with the capture reagentof under conditions suitable for forming a target analyte-capturereagent complex. The first specific binding partner is contacted with asecond specific binding member functionally associated with a seedparticle suitable for associating with a SERS substrate. The targetanalyte-capture reagent complex is then contacted with electromagneticradiation suitable for detecting a specific property associated with theanalyte-capture reagent complex by Raman spectroscopy.

In still another embodiment, the invention provides methods formultiplex detection of analytes in a sample by contacting targetanalytes in a sample under conditions suitable to form complexes with aset of capture reagents. In the case of a target analyte that is anon-nucleic acid, the capture reagent is conjugated with an Raman label.Each capture reagent can be conjugated to a unique Raman label, such asa Raman dye, associated with a unique optical signature. Followingcomplex formation and SERS activation of the SERS substrate, the uniqueoptical signatures are detected in a multiplex manner with a suitabledetection device. Since each specifically binding target analyte isbound to a specific capture reagent conjugated to a known Raman dye thatemits a distinguishable optical signature, such as a SERS signal,individual optical signatures detected from the constructs are thusassociated with the identity of a target analyte in the sample.

“Test sample,” as used herein, means the sample containing the targetanalyte to be detected and assayed using the present invention. The testsample can contain other components besides the target analyte, can havethe physical attributes of a liquid, or a solid, and can be of any sizeor volume, including for example, a moving stream of liquid. The testsample can contain any substances other than the target analyte as longas the other substances do no interfere with the binding of the targetanalyte with the capture reagent or the specific binding of the firstbinding member to the second binding member. Examples of test samplesinclude, but are not limited to: Serum, plasma, sputum, seminal fluid,urine, other body fluids, and environmental samples such as ground wateror waste water, soil extracts, air and pesticide residues.

The optical detection procedure or combination of optical detectionprocedures to be used will depend on the nature of the analytes, theseparation device or matrix, as well as the structure and properties ofthe capture reagents. The separated complexes can be detected by one ora combination of optical techniques selected from adsorption,reflection, polarization, refraction, fluorescence, Raman spectra, SERS,resonance light scattering, grating-coupled surface plasmon resonance,using techniques described herein and as known in the art.

The Raman active labels are selected from a Raman-active dye, aminoacid, nucleotide, or a combination thereof. Examples of Raman activeamino acids suitable for incorporation into the Raman-active tag includearginine, methionine, cysteine, and combinations thereof. Examples ofRaman-active nucleotides suitable for incorporation into theRaman-active tag include adenine, guanine and derivatives thereof. TheRaman-active labels are small molecules that are highly active inproducing a Raman signal and typically have a molecular weight of lessthan 1 kDa. Raman-active tags that meet these requirements include dyes(e.g., R6G, Tamra, Rox), amino acids (e.g., arginine, methionine,cysteine), nucleic acid bases (e.g., adenine, or guanine), or anycombination thereof. Naturally occurring or synthetic compounds havingthe above-described characteristics, such as suitable molecular weightand Raman characteristics, can also be used. The Raman-active tags canbe placed in any position along the molecular backbone and a singlebackbone can have more than one such tag. Raman signatures of themembers of the set can be adjusted by changing the type, number andrelative positions of the Raman-active tags along the backbone duringsynthesis of the molecular Raman codes.

The bound complexes including the Raman-active label is covered with athin layer of metal, as described herein, to enhance Raman signals fromthe complex. The metal layer, being in close proximity to the analyte,will produce SERS signals and the complete solid support can beirradiated with a single light source while SERS signals are collectedfrom the bound complexes, for example by SERS scanning. One or more SERSspectra obtained from a discrete site associates the capture reagentwith the presence of a particular target analyte in the sample oridentifies the capture reagent as having affinity (e.g., heretoforeunknown) for a molecule or complex in the sample.

Antibodies and receptors are non-limiting examples of the capturereagents attached to the discrete locations on the solid support.Nucleic acids, phage-displayed peptides, nucleic acids, aptamers,ligands, lectins, and combinations thereof can also be used as capturereagents in the invention methods. The sample is not necessarily a bodyfluid, although it may be, but can comprise any mixed pool of analytes,including proteins, gluco-proteins, lipid proteins, nucleic acids, virusparticles, polysaccharides, steroids, and combinations thereof. In oneaspect, the sample comprises a pool of body fluid of patients known orsuspected of having a particular disease.

The term “nucleic acid” is used broadly herein to mean a sequence ofdeoxyribonucleotides or ribonucleotides that are linked together by aphosphodiester bond. For convenience, the term “oligonucleotide” is usedherein to refer to a polynucleotide that is used as a primer or a probe.Generally, an oligonucleotide useful as a probe or primer thatselectively hybridizes to a selected nucleotide sequence is at leastabout 10 nucleotides in length, usually at least about 15 nucleotides inlength, for example between about 15 and about 50 nucleotides in length.

A nucleic acid can be RNA or can be DNA, which can be a gene or aportion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence,or the like, and can be single stranded or double stranded, as well as aDNA/RNA hybrid. In various embodiments, a polynucleotide, including anoligonucleotide (e.g., a probe or a primer) can contain nucleoside ornucleotide analogs, or a backbone bond other than a phosphodiester bond.In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotideor oligonucleotide also can contain nucleotide analogs, includingnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides. Such nucleotide analogs are well known in the artand commercially available, as are polynucleotides containing suchnucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234 (1994);Jellinek et al., Biochemistry 34:11363-11372 (1995); Pagratis et al.,Nature Biotechnol. 15:68-73 (1997).

The covalent bond linking the nucleotides of a nucleic acid generally isa phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond or any other bond known to those in the art asuseful for linking nucleotides to produce synthetic polynucleotides(see, for example, Tam et al., Nucl. Acids Res. 22:977-986 (1994); Eckerand Crooke, BioTechnology 13:351360 (1995)). The incorporation ofnon-naturally occurring nucleotide analogs or bonds linking thenucleotides or analogs can be particularly useful where thepolynucleotide is to be exposed to an environment that can contain anucleolytic activity, including, for example, a tissue culture medium orupon administration to a living subject, since the modifiedpolynucleotides can be less susceptible to degradation.

As used herein, the term “selective hybridization” or “selectivelyhybridize,” refers to hybridization under moderately stringent or highlystringent conditions such that a nucleotide sequence preferentiallyassociates with a selected nucleotide sequence over unrelated nucleotidesequences to a large enough extent to be useful in identifying theselected nucleotide sequence. It will be recognized that some amount ofnon-specific hybridization is unavoidable, but is acceptable providedthat hybridization to a target nucleotide sequence is sufficientlyselective such that it can be distinguished over the non-specificcross-hybridization, for example, at least about 2-fold more selective,generally at least about 3-fold more selective, usually at least about5-fold more selective, and particularly at least about 10-fold moreselective, as determined, for example, by an amount of labeledoligonucleotide that binds to target nucleic acid molecule as comparedto a nucleic acid molecule other than the target molecule, particularlya substantially similar (i.e., homologous) nucleic acid molecule otherthan the target nucleic acid molecule. Conditions that allow forselective hybridization can be determined empirically, or can beestimated based, for example, on the relative GC:AT content of thehybridizing oligonucleotide and the sequence to which it is tohybridize, the length of the hybridizing oligonucleotide, and thenumber, if any, of mismatches between the oligonucleotide and sequenceto which it is to hybridize (see, for example, Sambrook et al.,“Molecular Cloning: A laboratory manual (Cold Spring Harbor LaboratoryPress 1989)).

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42EC (moderate stringency conditions); and0.1×SSC at about 68EC (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

As used herein, the term “antibody” is used in its broadest sense toinclude polyclonal and monoclonal antibodies, as well as antigen bindingfragments of such antibodies. An antibody useful in a method of theinvention, or an antigen binding fragment thereof, is characterized, forexample, by having specific binding activity for an epitope of ananalyte. Alternatively, as explained below, the analyte can be the probeantibody, particularly in embodiments of the invention methods whereinantibodies used as probes (e.g. active agents) are exposed to bodyfluids to screen a set of antibodies for utility as drug candidates.

The antibody, for example, includes naturally occurring antibodies aswell as non-naturally occurring antibodies, including, for example,single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains (see Huse et al., Science246:1275-1281 (1989)). These and other methods of making, for example,chimeric, humanized, CDR-grafted, single chain, and bifunctionalantibodies are well known to those skilled in the art (Winter andHarris, Immunol. Today 14:243-246, 1993; Ward et al., Nature341:544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual(Cold Spring Harbor Laboratory Press, 1988); Hilyard et al., ProteinEngineering: A practical approach (IRL Press 1992); Borrabeck, AntibodyEngineering, 2d ed. (Oxford University Press 1995)). Monoclonalantibodies suitable for use as probes may also be obtained from a numberof commercial sources. Such commercial antibodies are available againsta wide variety of targets. Antibody probes can be conjugated tomolecular backbones using standard chemistries, as discussed below.

The term “binds specifically” or “specific binding activity,” when usedin reference to an antibody means that an interaction of the antibodyand a particular epitope has a dissociation constant of at least about1×10⁻⁶, generally at least about 1×10⁻⁷, usually at least about 1×10⁻⁸,and particularly at least about 1×10⁻⁹ or 1×10⁻¹⁰ or less. As such, Fab,F(ab′)₂, Fd and Fv fragments of an antibody that retain specific bindingactivity for an epitope of an antigen, are included within thedefinition of an antibody.

In the context of the invention, the term “ligand” denotes a naturallyoccurring specific binding partner of a receptor, a syntheticspecific-binding partner of a receptor, or an appropriate derivative ofthe natural or synthetic ligands. The determination and isolation ofligands is well known in the art (Lerner, Trends Neurosci. 17:142-146,1994). As one of skill in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

In certain aspects, the invention pertains to methods for detecting ananalyte in a sample. By “analyte” is meant any molecule or compound forwhich a probe can be found. An analyte can be in the solid, liquid,gaseous or vapor phase. By “gaseous or vapor phase analyte” is meant amolecule or compound that is present, for example, in the headspace of aliquid, in ambient air, in a breath sample, in a gas, or as acontaminant in any of the foregoing. It will be recognized that thephysical state of the gas or vapor phase can be changed by pressure,temperature as well as by affecting surface tension of a liquid by thepresence of or addition of salts etc.

The analyte can be a molecule found directly in a sample such as a bodyfluid from a host. The sample can be examined directly or can bepretreated to render the analyte more readily detectable. Furthermore,the analyte of interest can be determined by detecting an agentprobative of the analyte of interest such as a specific binding pairmember complementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay. The body fluid can be, for example, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, and the like.

As used herein, the term “colloid” refers to metal ions suspended in aliquid, usually water. Typical metals contemplated for use in inventionmetal colloids and to from nanoparticles include the transparent metals,for example, silver, gold, platinum, aluminum, and the like.

To enhance the Raman spectra produced by Raman-active substrates, a thinlayer of a transparent metal, wherein the layer has a roughened surface,is deposited over the upper layer of the substrate and/or the boundcomplexes thereon. The roughness features are on the order of tens ofnanometers; small, compared to the wavelength of the incident excitationradiation. The small size of the particles allows the excitation of themetal particle's surface plasmon to be localized on the particle. Metalroughness features at the metal surface can be developed in a number ofways; for example; vapor deposition of metal particles or application ofmetal colloids onto the upper layer of the biosensor. Since the surfaceelectrons of the metal are confined to the particle, whose size issmall, the plasmon excitation is also confined to the roughness feature.The resulting electromagnetic field of the plasmon is very intense,greatly enhancing the SERS signal as compared to a Raman signal.

It has been estimated that only 1 in 10 analyte molecules inelasticallyscatter in Raman Spectroscopy. However, in embodiments of the inventionmethods wherein the intensity of Raman signal from a scattering moleculeis greatly enhanced under SERS conditions, low concentrations of aRaman-active analyte can be detected at concentrations as low as pico-and femto-molar. In some circumstances, the invention methods can beused to detect the presence of a single analyte molecule in a complexbiological sample, such as blood serum, by depositing a thin layer of atransparent metal so as to be in contact with the bound complexescontaining a Raman label. Gold, silver, copper and aluminum are thetransparent metals most useful for this technique.

A roughened metal surface can be produced using one of several methods.The term “a thin metal layer” as used herein means a metal layerdeposited by chemical vapor deposition over the bound complexescontaining a Raman label. Alternatively, a thin metal layer means alayer of nanoparticles formed by subjecting a colloidal solution ofmetal cations to reducing conditions to form metal nanoparticles insitu. In some embodiments, the nanoparticles will contain the boundcomplexes. Alternatively, seed particles, for example attached to theRaman codes, can precipitate formation of the nanoparticles from a metalcolloid solution. In this context, “thin” means having a thickness ofabout one-half the wavelength of the irradiating light source (usually alaser) to achieve the benefit of SERS, for example from about 15 nm toabout 500 nm, such as about 100 nm to about 200 nm.

The “analytes”, as the term is used herein, includes nucleic acids,proteins, peptides, lipids, carbohydrates, glycolipids, glycoproteins orany other potential target for which a specific probe can be prepared.As discussed above, antibody or aptamer probes can be incorporated intothe invention active molecular Raman codes and used to identify anytarget for which an aptamer or antibody can be prepared. The presence ofmultiple analytes in a sample can be assayed simultaneously, since eachmember of a set can be distinguishably labeled and detected.Quantification of the analyte can be performed by standard techniques,well known in spectroscopic analysis. For example, the amount of analytebound to an invention Raman probe construct can be determined bymeasuring the signal intensity produced and comparison to a calibrationcurve prepared from known amounts of similar Raman probe constructstandards. Such quantification methods are well within the routine skillin the art.

A “substrate” as the term is used herein, includes such well knowndevices as chips or microtiter plates, may comprise a patterned surfacecontaining individual discrete sites that can be treated as describedherein bind to individual analytes or types of analytes. Alternatively,in embodiments wherein the probe Raman construct is attached to thesubstrate, a correlation between the location of an individual site onthe array with the Raman code or probe located at that particular sitecan be made.

Array compositions may include at least a surface with a plurality ofdiscrete substrate sites. The size of the array will depend on the enduse of the array. Arrays containing from about 2 to many millions ofdifferent discrete substrate sites can be made. Generally, the arraywill comprise from two to as many as a billion or more such sites,depending on the size of the surface. Thus, very high density, highdensity, moderate density, low density or very low density arrays can bemade. Some ranges for very high-density arrays are from about 10,000,000to about 2,000,000,000 sites per array. High-density arrays range fromabout 100,000 to about 10,000,000 sites. Moderate density arrays rangefrom about 10,000 to about 50,000 sites. Low-density arrays aregenerally less than 10,000 sites. Very low-density arrays are less than1,000 sites.

The sites comprise a pattern, i.e. a regular design or configuration, orcan be randomly distributed. A regular pattern of sites can be used suchthat the sites can be addressed in an X-Y coordinate plane. The surfaceof the substrate can be modified to allow attachment of analytes atindividual sites. Thus, the surface of the substrate can be modifiedsuch that discrete sites are formed. In one embodiment, the surface ofthe substrate can be modified to contain wells, i.e. depressions in thesurface of the substrate. This can be done using a variety of knowntechniques, including, but not limited to, photolithography, stampingtechniques, molding techniques and microetching techniques. As will beappreciated by those in the art, the technique used will depend on thecomposition and shape of the substrate. Alternatively, the surface ofthe substrate can be modified to contain chemically derived sites thatcan be used to attach analytes or probes to discrete locations on thesubstrate. The addition of a pattern of chemical functional groups, suchas amino groups, carboxy groups, oxo groups and thiol groups can be usedto covalently attach molecules containing corresponding reactivefunctional groups or linker molecules.

Biological “analytes” may comprise naturally occurring proteins orfragments of naturally occurring proteins. Thus, for example, cellularextracts containing proteins, or random or directed digests ofproteinaceous cellular extracts, can be used. In this way libraries ofprocaryotic and eukaryotic proteins can be made for screening thesystems described herein. For example libraries of bacterial, fungal,viral, and mammalian proteins can be generated for screening purposes.

Methods for oligonucleotide synthesis are well known in the art and anysuch known method can be used. For example, oligonucleotides can beprepared using commercially available oligonucleotide synthesizers(e.g., Applied Biosystems, Foster City, Calif.). Nucleotide precursorsattached to a variety of tags can be commercially obtained (e.g.Molecular Probes, Eugene, Oreg.) and incorporated into oligonucleotidesor polynucleotides. Alternatively, nucleotide precursors can bepurchased containing various reactive groups, such as biotin,diogoxigenin, sulfhydryl, amino or carboxyl groups. Afteroligonucleotide synthesis, tags can be attached using standardchemistries. Oligonucleotides of any desired sequence, with or withoutreactive groups for tag attachment, may also be purchased from a widevariety of sources (e.g., Midland Certified Reagents, Midland, Tex.).

The metal required for achieving a suitable SERS signal is inherent inthe COIN, and a wide variety of Raman-active organic compounds can beincorporated into the particle. Indeed, a large number of unique Ramansignatures can be created by employing nanoparticles containingRaman-active organic compounds of different structures, mixtures, andratios. Thus, the methods described herein employing COINs are usefulfor the simultaneous detection of many analytes in a sample, resultingin rapid qualitative analysis of the contents of “profile” of a bodyfluid. In addition, since many COINs can be incorporated into a singlenanoparticle, the SERS signal from a single COIN particle is strongrelative to SERS signals obtained from Raman-active materials that donot contain the nanoparticles described herein as COINs. This situationresults in increased sensitivity compared to Raman-techniques that donot utilize COINs.

COINs are readily prepared for use in the invention methods usingstandard metal colloid chemistry. The preparation of COINs also takesadvantage of the ability of metals to adsorb organic compounds. Indeed,since Raman-active organic compounds are adsorbed onto the metal duringformation of the metallic colloids, many Raman-active organic compoundscan be incorporated into the COIN without requiring special attachmentchemistry.

In general, the COINs used in the invention methods are prepared asfollows. An aqueous solution is prepared containing suitable metalcations, a reducing agent, and at least one suitable Raman-activeorganic compound. The components of the solution are then subject toconditions that reduce the metallic cations to form neutral, colloidalmetal particles. Since the formation of the metallic colloids occurs inthe presence of a suitable Raman-active organic compound, theRaman-active organic compound is readily adsorbed onto the metal duringcolloid formation. This simple type of COIN is referred to as type ICOIN. Type I COINs can typically be isolated by membrane filtration. Inaddition, COINs of different sizes can be enriched by centrifugation.

In alternative embodiments, the COINs can include a second metaldifferent from the first metal, wherein the second metal forms a layeroverlying the surface of the nanoparticle. To prepare this type ofSERS-active nanoparticle, type I COINs are placed in an aqueous solutioncontaining suitable second metal cations and a reducing agent. Thecomponents of the solution are then subject to conditions that reducethe second metallic cations so as to form a metallic layer overlying thesurface of the nanoparticle. In certain embodiments, the second metallayer includes metals, such as, for example, silver, gold, platinum,aluminum, and the like. This type of COIN is referred to as type IICOINs. Type II COINs can be isolated and or enriched in the same manneras type I COINs. Typically, type I and type II COINs are substantiallyspherical and range in size from about 20 nm to 60 nm. The size of thenanoparticle is selected to be about one-half the wavelength of lightused to irradiate the COINs during detection.

Typically, organic compounds are attached to a layer of a second metalin type II COINs by covalently attaching organic compounds to thesurface of the metal layer Covalent attachment of an organic layer tothe metallic layer can be achieved in a variety ways well known to thoseskilled in the art, such as for example, through thiol-metal bonds. Inalternative embodiments, the organic molecules attached to the metallayer can be crosslinked to form a molecular network.

The COIN(s) used in the invention methods can include cores containingmagnetic materials, such as, for example, iron oxides, and the like.Magnetic COINs can be handled without centrifugation using commonlyavailable magnetic particle handling systems. Indeed, magnetism can beused as a mechanism for separating biological targets attached tomagnetic COIN particles tagged with particular biological probes.

As used herein, “Raman-active organic compound” refers to an organicmolecule that produces a unique SERS signature in response to excitationby a laser. A variety of Raman-active organic compounds are contemplatedfor use as components in COINs. In certain embodiments, Raman-activeorganic compounds are polycyclic aromatic or heteroaromatic compounds.Typically the Raman-active organic compound has a molecular weight lessthan about 300 Daltons.

Additional, non-limiting examples of Raman-active organic compoundsuseful in COINs include TRIT (tetramethyl rhodamine isothiol), NBD(7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blueviolet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine,biotin, digoxigenin, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxy rhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino phthalocyanines, azomethines, cyanines,xanthines, succinylfluoresceins, aminoacridine, and the like. These andother Raman-active organic compounds can be obtained from commercialsources (e.g., Molecular Probes, Eugene, Oreg.).

In certain embodiments, the Raman-active compound is adenine, adenine,4-amino-pyrazolo(3,4-d)pyrimidine, 2-fluoroadenine, N6-benzolyadenine,kinetin, dimethyl-allyl-amino-adenine, zeatin, bromo-adenine,8-aza-adenine, 8-azaguanine, 6-mercaptopurine,4-amino-6-mercaptopyrazolo(3,4-d)pyrimidine, 8-mercaptoadenine, or9-amino-acridine 4-amino-pyrazolo(3,4-d)pyrimidine, or 2-fluoroadenine.In one embodiment, the Raman-active compound is adenine.

When “fluorescent compounds” are incorporated into COINs, thefluorescent compounds can include, but are not limited to, dyes,intrinsically fluorescent proteins, lanthanide phosphors, and the like.Dyes useful for incorporation into COINs include, for example, rhodamineand derivatives, such as Texas Red, ROX (6-carboxy-X-rhodamine),rhodamine-NHS, and TAMRA (5/6-carboxytetramethyl rhodamine NHS);fluorescein and derivatives, such as 5-bromomethyl fluorescein and FAM(5′-carboxyfluorescein NHS), Lucifer Yellow, IAEDANS, 7-Me₂,N-coumarin-4-acetate, 7-OH-4-CH₃-coumarin-3-acetate,7-NH₂-4CH₃-coumarin-3-acetate (AMCA), monobromobimane, pyrenetrisulfonates, such as Cascade Blue, andmonobromotrimethyl-ammoniobimane.

Exemplary uses for the methods described herein is to detect a targetnucleic acid. Such a method is useful, for example, for detection of asingle nucleotide polymorphism (SNP), for detection of infectious agentswithin a clinical sample, detection of an amplification product derivedfrom genomic DNA or RNA or message RNA, or detection of a gene (cDNA)insert within a clone. For certain methods aimed at detection of atarget polynucleotide, an oligonucleotide probe is synthesized usingmethods known in the art.

In the practice of the present invention, the Raman spectrometer can bepart of a detection unit designed to detect and quantify Raman signalsof the present invention by Raman spectroscopy. Methods for detection ofRaman labeled analytes, for example nucleotides, using Ramanspectroscopy are known in the art. (See, e.g., U.S. Pat. Nos. 5,306,403;6,002,471; and 6,174,677). Variations on surface enhanced Ramanspectroscopy (SERS), surface enhanced resonance Raman spectroscopy(SERRS) and coherent anti-Stokes Raman spectroscopy (CARS) have beendisclosed.

A non-limiting example of a Raman detection unit is disclosed in U.S.Pat. No. 6,002,471. An excitation beam is generated by either afrequency doubled Nd:YAG laser at 532 nm wavelength or a frequencydoubled Ti:sapphire laser at 365 nm wavelength. Pulsed laser beams orcontinuous laser beams can be used. The excitation beam passes throughconfocal optics and a microscope objective, and is focused onto the flowpath and/or the flow-through cell. The Raman emission light is collectedby the microscope objective and the confocal optics and is coupled to amonochromator for spectral dissociation. The confocal optics includes acombination of dichroic filters, barrier filters, confocal pinholes,lenses, and mirrors for reducing the background signal. Standard fullfield optics can be used as well as confocal optics. The Raman emissionsignal is detected by a Raman detector, that includes an avalanchephotodiode interfaced with a computer for counting and digitization ofthe signal.

Another example of a Raman detection unit is disclosed in U.S. Pat. No.5,306,403, including a Spex Model 1403 double-grating spectrophotometerwith a gallium-arsenide photomultiplier tube (RCA Model C31034 or BurleIndustries Model C3103402) operated in the single-photon counting mode.The excitation source includes a 514.5 nm line argon-ion laser fromSpectraPhysics, Model 166, and a 647.1 nm line of a krypton-ion laser(Innova 70, Coherent).

Alternative excitation sources include a nitrogen laser (Laser ScienceInc.) at 337 nm and a helium-cadmium laser (Liconox) at 325 nm (U.S.Pat. No. 6,174,677), a light emitting diode, an Nd:YLF laser, and/orvarious ions lasers and/or dye lasers. The excitation beam can bespectrally purified with a bandpass filter (Corion) and can be focusedon the flow path and/or flow-through cell using a 6× objective lens(Newport, Model L6X). The objective lens can be used to both excite theRaman-active probe constructs and to collect the Raman signal, by usinga holographic beam splitter (Kaiser Optical Systems, Inc., Model HB647-26N18) to produce a right-angle geometry for the excitation beam andthe emitted Raman signal. A holographic notch filter (Kaiser OpticalSystems, Inc.) can be used to reduce Rayleigh scattered radiation.Alternative Raman detectors include an ISA HR-320 spectrograph equippedwith a red-enhanced intensified charge-coupled device (RE-ICCD)detection system (Princeton Instruments). Other types of detectors canbe used, such as Fourier-transform spectrographs (based on Michaelsoninterferometers), charged injection devices, photodiode arrays, InGaAsdetectors, electronmultiplied CCD, intensified CCD and/orphototransistor arrays.

Any suitable form or configuration of Raman spectroscopy or relatedtechniques known in the art can be used for detection of the targetcomplex of the present invention, including but not limited to normalRaman scattering, resonance Raman scattering, surface enhanced Ramanscattering, surface enhanced resonance Raman scattering, coherentanti-Stokes Raman spectroscopy (CARS), stimulated Raman scattering,inverse Raman spectroscopy, stimulated gain Raman spectroscopy,hyper-Raman scattering, molecular optical laser examiner (MOLE) or Ramanmicroprobe or Raman microscopy or confocal Raman microspectrometry,three-dimensional or scanning Raman, Raman saturation spectroscopy, timeresolved resonance Raman, Raman decoupling spectroscopy or UV-Ramanmicroscopy.

In certain aspects of the invention, a system for detecting the targetcomplex of the present invention includes an information processingsystem. An exemplary information processing system may incorporate acomputer that includes a bus for communicating information and aprocessor for processing information. In one embodiment of theinvention, the processor is selected from the Pentium® family ofprocessors, including without limitation the Pentium® II family, thePentium® III family and the Pentium® 4 family of processors availablefrom Intel Corp. (Santa Clara, Calif.). In alternative embodiments ofthe invention, the processor can be a Celeron®, an Itanium®, or aPentium Xeon® processor (Intel Corp., Santa Clara, Calif.). In variousother embodiments of the invention, the processor can be based on Intel®architecture, such as Intel® IA-32 or Intel® IA-64 architecture.Alternatively, other processors can be used. The information processingand control system may further comprise any peripheral devices known inthe art, such as memory, display, keyboard and/or other devices.

In particular examples, the detection unit can be operably coupled tothe information processing system. Data from the detection unit can beprocessed by the processor and data stored in memory. Data on emissionprofiles for various Raman labels may also be stored in memory. Theprocessor may compare the emission spectra from the target complexes inthe flow path and/or flow-through cell to identify the Raman-activemoiety in complexed with the target analyte or the capture reagent. Theinformation processing system may also perform standard procedures suchas subtraction of background signals or comparison of signals fromdifferent samples.

While certain methods of the present invention can be performed underthe control of a programmed processor, in alternative embodiments of theinvention, the methods can be fully or partially implemented by anyprogrammable or hardcoded logic, such as Field Programmable Gate Arrays(FPGAs), TTL logic, or Application Specific Integrated Circuits (ASICs).Additionally, the disclosed methods can be performed by any combinationof programmed general purpose computer components and/or custom hardwarecomponents.

Following the data gathering operation, the data will typically bereported to a data analysis operation. To facilitate the analysisoperation, the data obtained by the detection unit will typically beanalyzed using a digital computer such as that described above.Typically, the computer will be appropriately programmed for receipt andstorage of the data from the detection unit as well as for analysis andreporting of the data gathered.

In certain embodiments of the invention, custom designed softwarepackages can be used to analyze the data obtained from the detectionunit. In alternative embodiments of the invention, data analysis can beperformed using an information processing system and publicly availablesoftware packages.

Exemplary Raman labels are provided below in Table 1.

TABLE 1 Raman labels selection Abbreviation Name Structure AAD (AA)8-Aza-Adenine

BZA (BA) N-Benzoyladenine

APP 4-Amino-pyrazolo [3,4-d]pyrimidine

ZEN Zeatin

MBL Methylene Blue

AMA (AM) 9-Amino-acridine

EBR Ethidium Bromide

BMB Bismarck Brown Y

THN Thionin acetate

DAH 3,6-Diaminoacridine

AIC 4-Amino-5-imidazole- carboxamide hydrochloride

DII 1,3-Diiminoisoindoline

R6G Rhodamine 6G

CRV Crystal Violet

BFU Basic Fuchsin

NBA N-Benzyl-aminopurine

MBI 2-Mercapto-benzimidazole (MBI)

CYP 6-Cyanopurine

ANB Aniline Blue diammonium salt

ACA N-[(3-(Anilinomethylene)- 2-chloro-1-cyclohexen-1-yl)methylene]aniline mono- hydrochloride

ATT O-(7-Azabenzotriazol-1-yl)- N,N,N′,N′- tetramethyluroniumhexafluorophosphate

AMF 9-Aminofluorene hydrochloride

BBL Basic Blue

DDA 1,8-Diamino-4,5- dihydroxyanthraquinone

PFV Proflavine hemisulfate salt hydrate

VRA Variamine Blue RT Salt

ABZ 2-Amino-benzothiazole

MEL Melamine

PPN 3-(3-Pyridylmethyl amino)propionitrile

SSD Silver(I) sulfadiazine

AMPT 4-Amino6-Mercaptopyrazolo [3,4-d]pyrimidine

APU 2-Am-Purine

ATH Adenine Thiol

FAD F-Adenine

MCP 6-Mercaptopurine

AMP 4-Amino-6-mercaptopyrazolo [3,4-d]pyrimidine

R110 Rhodamine 110

DAB 4-([4-(Dimethylamino) phenyl]azo)benzoic acid

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1-26. (canceled)
 27. A biological target complex comprising: a targetanalyte bound to a first specific binding member; a second specificbinding member that binds to the first specific binding member forming atarget complex, wherein the second specific binding member comprises aseed particle suitable for catalyzing the formation of a surfaceenhanced Raman scattering (SERS) substrate, wherein the SERS substrateis suitable to be activated to provide a SERS effect; a capture reagentbound to a solid substrate, wherein the capture reagent comprises aRaman label, wherein the target analyte binds to the capture reagentforming a biological target complex; and a layer of roughened metal overthe substrate or the biological target complex.
 28. The biologicaltarget complex of claim 27, wherein the layer of roughened metalcomprises roughness features on the order of tens of nanometers.
 29. Thebiological target complex of claim 28, wherein plasmon excitation due toelectromagnetic irradiation of the biological target complex is confinedto the roughness features.
 30. The biological target complex of claim29, wherein the layer of roughened metal comprises a thickness ofapproximately one-half the wavelength of the electromagneticirradiation.
 31. The biological target complex of claim 27, wherein thelayer is transparent.
 32. The biological target complex of claim 27,wherein the layer of roughened metal comprises gold, silver, copper, oraluminum.
 33. The biological target complex of claim 27, wherein thetarget analyte is a DNA, RNA, polypeptide, antibody, antigen,carbohydrate or small molecule.
 34. The biological target complex ofclaim 27, wherein the capture reagent is a DNA, RNA, polypeptide,antibody, antigen, carbohydrate or small molecule.
 35. The biologicaltarget complex of claim 27, wherein the first or second specific bindingmember is a DNA, RNA, antibody, antigen, polypeptide or carbohydrate.36. The biological target complex of claim 27, wherein the targetanalyte further comprises an ancillary specific binding member.
 37. Amethod comprising: a) providing a target analyte bound to a firstspecific binding member; b) providing a capture reagent bound to a solidsubstrate, wherein the capture reagent comprises a Raman label; c)contacting the target analyte of a) with the capture reagent of (b)under conditions suitable for forming a target analyte-capture reagentcomplex; d) contacting, prior to, concurrently with, or subsequent to c)the first specific binding partner with a second specific binding memberfunctionally associated with a seed particle suitable for associatingwith a SERS substrate, wherein the first specific binding member bindsto the second specific binding member; and e) coating either thesubstrate or the target analyte-capture reagent complex with a layer ofroughened metal; f) contacting the target analyte-capture reagentcomplex with electromagnetic radiation suitable for detecting a specificproperty associated with the analyte-capture reagent complex by Ramanspectroscopy.
 38. The method of claim 37, wherein the layer of roughenedmetal comprises roughness features on the order of tens of nanometers.39. The method of claim 37, wherein plasmon excitation due to theelectromagnetic irradiation is confined to the roughness features. 40.The method of claim 37, wherein the layer comprises a thickness ofapproximately one-half the wavelength of the electromagneticirradiation.
 41. The method of claim 37, wherein the layer of roughenedmeta is transparent.
 42. The method of claim 37, wherein the layer ofroughened metal comprises gold, silver, copper, or aluminum.
 43. Themethod of claim 37, comprising forming the layer of roughened metal byvapor deposition of metal particles or application of metal colloids.44. The method of claim 43, wherein application of metal colloidscomprises subjecting a colloidal solution of metal cations to reducingconditions to form metal nanoparticles in situ.
 45. The method of claim43, wherein application of metal colloids comprises using seed particleto precipitate nanoparticles from a metal colloid solution.
 46. Themethod of claim 45, wherein the seed particle is selected from the groupconsisting of gold, Ag, Cu, Pt, Ag/Au, Pt/Au, Cu/Au coreshell and alloyparticles.
 47. The method of claim 37, further comprising detecting asingle analyte molecule.
 48. A system comprising: biological targetcomplex comprising a target analyte bound to a first specific bindingmember, a second specific binding member that binds to the firstspecific binding member forming a target complex, the second specificbinding member comprising a seed particle suitable for catalyzing theformation of a surface enhanced Raman scattering (SERS) substrate, acapture reagent bound to a solid substrate, the capture reagentcomprising a Raman label, wherein the target analyte binds to thecapture reagent forming the biological target complex and a layer ofroughened metal over the substrate or the biological target complex; andan electromagnetic radiation source.
 49. The system of claim 48, furthercomprising a Raman detection unit.
 50. The system of claim 48, whereinthe electromagnetic radiation source comprises a frequency doubledNd:YAG laser, a frequency doubled Ti:sapphire laser, a nitrogen laser, ahelium-cadmium laser a light emitting diode, an Nd:YLF laser, ionlasers, or dye lasers.
 51. The system of claim 48, wherein the radiationsource is either pulsed or continuous.
 52. The system of claim 48,further comprising confocal optics and a microscope objective.
 53. Thesystem of claim 48, further comprising a flow through cell.
 54. Thesystem of claim 48, further comprising a monochromator.
 55. The systemof claim 52, wherein the confocal optics comprises one or more ofdichroic filters, barrier filters, holographic notch filters, confocalpinholes, lenses, and mirrors.
 56. The system of claim 49, wherein theRaman detection unit comprises an avalanche photodiode interfaced with acomputer.
 57. The system of claim 56, wherein the Raman detection unitis configured to count and digitize a signal.
 58. The system of claim49, wherein the Raman detection unit comprises a double-gratingspectrophotometer with a gallium-arsenide photomultiplier tube,Fourier-transform spectrographs, charged injection devices, photodiodearrays, InGaAs detectors, electronmultiplied CCD, intensified CCD orphototransistor arrays.
 59. The system of claim 49, wherein the Ramandetection unit comprises a red-enhanced intensified charge-coupleddevice (RE-ICCD) detection system.